Royal jelly (RJ) is a honeybee secretion, has numerous medicinal properties in particular antioxidant activities. Ischemia/reperfusion (I/R) is one of the main challenges in acute kidney damage. This study was designed to assess the anti-inflammatory and protective effects of RJ against I/R-induced renal disorders. Forty male rats were randomly divided into four groups (n = 10) as sham (0.9% saline) group, I/R group, RJ group (treated for 15 consecutive days by gavage with 300 mg/kg/day RJ), and I/R + RJ group that were pretreated for 15 consecutive days by gavage with 300 mg/kg/day of RJ. The I/R-induced renal inflammation was evaluated by determining leukocyte infiltration and mRNA expression level of intercellular adhesion molecule-1 and tumor necrotic factor-alpha (TNF-α). Antioxidant capacity of kidneys and thiobarbituric acid reactive species was measured in kidneys for the evaluation of oxidative stress. In addition, the diameter of renal glomeruli, kidney function indicators, and serum nitrite oxide (NO) levels was determined. The I/R increased the completely measured parameters, except the tissue ferric reducing/antioxidant power (FRAP) level, which was decreased compared to the sham group (P < 0.05). However, pretreatment with RJ reduced significantly blood urea nitrogen, kidney malondialdehyde, creatinine, glomerular diameter, leukocyte infiltration, levels of TNF-α, adhesion molecule-1 expression, and NO and increased tissue FRAP compared to the I/R group (P < 0.05). It seems that RJ administration improved I/R-induced acute kidney injury.

Introduction of antioxidant compounds can be an appropriate strategy to decrease the oxidative stress-induced damage.[1] Royal jelly (RJ) is a mixture of secretions of mandibular glans of honeybees that plays a key role in neonatal growth, completion of special sexual features, and long life of the queen. Hypopharyngeal glands in honeybees play a crucial role in the beehive development, especially queen by producing RJ.[2] Bees feed on RJ in the first 3 days, and their diet is changed afterward, while the queen always feeds on RJ.[3] RJ contains a wide range of chemical compounds such as proteins (17%–45% dry weight), royalism (with potent antibacterial properties), apisimin (stimulating the proliferation of monocytes in human), Jelleines (with antimicrobial activity), and lipids, fatty acids, carbohydrates, vitamins (Group B vitamins, thiamine, riboflavin, pentatonic acid, niacin, folic acid, and biotin).[4] The antioxidant activity of RJ inhibits oxidative stress and lipid peroxidation and protects DNA against oxidative stress.[5] Kanbur et al. reported that mice fed with RJ had a higher level of 8-hydroxy-2-decenoic acid (oxidative stress marker) in their liver and blood serum, with increased average longevity.[6] Further, Karadeniz et al. showed that the administration of RJ decreased kidney toxicity and oxidative stress induced by cisplatin compounds.[7] Ischemia/reperfusion (I/R) is one of the major causes for acute kidney injury (AKI).[8] AKI happens in 5% of hospitalized patients and is related to 30%–50% of admissions to the intensive care unit.[9] Pathophysiologically, one of the challenges in the managing of AKI is inflammation, which also leads to the progress of renal conflicts. Renal ischemia and nephrotoxins are the main causes of AKI.[10] I/R have toxic effects, and RJ has numerous beneficial properties, especially antioxidant properties. Further, no study has ever investigated the effects of RJ on the I/R-induced impairments in the kidney tissue. Hence, the purpose of this study is to evaluate the anti-inflammatory, kidneys antioxidant capacity, functional and morphological effects of RJ due to the beneficial and multiple properties on AKI induced by I/R in male rats has not been clarified yet.

Materials and Methods

Animals

In this experimental study, 40 male Wistar rats (weighing 220–250 g) were purchased from the Pasteur Institute and transferred to the animal house in medical school. During the study, the animals were kept under standard conditions (12 h light/12 h dark and 22°C ± 2°C), in special cages and on a straw bed. Water and food were provided ad libitum to the animals. Standard food and treated municipal water were used to feed the animals. All investigations conformed to the ethical and humane principles of research and were approved by the Ethics Committee of Kermanshah University of Medical Sciences (ethics certificate No. 97507).[1]

Study groups and treatment

Forty male rats were randomly assigned into four groups and 10 rats were placed in each group. The first group was the sham group that received normal saline 0.9% equivalent to the number of experimental groups. In this group, all surgical procedures were performed, but the renal arteries were not occluded. In the second group, the I/R group, 30 min after saline injection, animals underwent a bilateral renal ischemia for 30 min followed by 24 h of reperfusion. The third group was RJ group, in this group, animals were treated orally for 15 consecutive days with 300 mg/kg/day RJ. The fourth group was RJ + I/R group, in this group, 30 min after RJ (300 mg/kg) administration, rats underwent a bilateral renal ischemia for 30 min followed by 24 h of reperfusion. All the rats received 100 International Unit of heparin (Merck) intraperitoneally and 30 min preoperatively.[7],[8]

Surgery and induction of renal ischemia/reperfusion

After anesthesia with intraperitoneally injection of ketamine HCl (100 mg/kg) and Xylazine (10 mg/kg) (Merck) and followed by 100 units of heparin (Merck) to prevent intravascular blood clotting, in the linea alba, a longitudinal incision was created by means of electrocautery (Surgistat-USA), both renal arteries were clamped at the same time by a nontraumatic microvascular clamp (Roboz Surgical Instruments-USA) for 30 min, and then, they released to permit reperfusion. Throughout the surgery, the body temperature was controlled within the range of 37°C ± 1°C through a rectal probe connected to a thermistor. Rats were then dissected, their kidneys were removed, and the left kidneys were separated longitudinally into two halves. One-half was processed for morphometric study and leukocyte infiltration. The other half was used to measure the number of intercellular adhesion molecule-1 (ICAM-1) and tumor necrotic factor-alpha (TNF-α) mRNA expression. The right kidneys were used for oxidative stress measurements.[9]

Kidney function assay

Blood samples were collected from the animal's abdominal aorta, after 24-h reperfusion period. The blood samples were incubated at 37°C for 15 min and centrifuged at 4000 rpm for 15 min to acquire the serum. The serum samples were kept in a −20°C freezer. Plasma samples were assayed for concentrations of plasma creatinine and blood urea nitrogen (BUN) using an autoanalyzer (RA 1000; Technicon Instruments; USA).[11]

Measuring the diameter of renal glomeruli and rate of leukocyte infiltration

Samples were fixed by 10% formalin solution and washing. The process of tissue preparation was based on the conventional histology method (paraffin method), i.e., passing the samples from ascending alcohols for dehydration, passing through the mesh for clarification and removal of turbidity, and ultimately passing paraffin for infiltration and filling the vacuum created at the pores of the fat. All of the process, steps were performed using Automatic Tissue Processors. After molding each sample, serial sections were prepared by a microtome (Leica RM 2125, Leica Microsystems Nussloch GmbH; Germany) (slices with 5-μm diameter) from the tissue. Among all sections, about 10 samples were selected for staining. Therefore, 10 slides were prepared from the kidneys of each rat. An optical microscope using a hematoxylin and eosin staining method stained the prepared slices. The diameter of glomeruli was examined under a microscope (Olympus BX-51T-32E01, Japan) linked to a DP12 camera with 3.34-million pixel resolution and Olysia Bio-software (Olympus Optical; Japan). To determine the amount of leukocyte infiltration, the number of leukocytes in 20 microscopic fields each consisting of 0.14 mm2 was counted, averaged, and used to estimate the quantity for each square millimeter.[9]

Real-time polymerase chain reaction assay

To assess inflammation, TNF-α and ICAM-1 mRNA expression levels were measured, following the evaluation rate of leukocyte infiltration. Samples were frozen in liquid nitrogen and stored in a freezer at −80°C pending examination. In the initial step, RNA was extracted from the kidney tissue using the RNeasy mini kit (Qiagen co) according to the manufacturer's instructions. By DNase set kit, the extracted DNA samples were treated to eliminate the genomic DNA. The cDNA version was produced from the RNA extracted from the previous step by means of RevertAid™First Strand cDNA Synthesis Kit. The expression level of the given gene was measured through glyceraldehyde 3-phosphate dehydrogenase primer as endogenous control by Maxima SYBR Green/Rox quantitative polymerase chain reaction master mix (Fermentas co) through Comparative Ct technique. First, a denaturation at 95°C in 10 min, a denaturation at 95°C in 15 s, and annealing extension at 60°C in 1 min with 40 cycles were performed and then melt curves analysis (60°C → 95°C increment of 0.3°C) was used to determine the melting temperature of specific amplification products and primer dimers (StepOnePlus, Applied biosystem). The sequence of primers used is shown in [Table 1].[12]

In order to assess oxidative stress, the thiobarbituric acid reactive species were measured using malondialdehyde (MDA) as the last product of lipid peroxidation in kidney tissue by colorimetric analyze. In brief, 1400 μl of acetic acid (Sigma, USA), 1400 μl of TBA (Sigma, USA), and 1400 μl of sodium dodecyl sulfate (Sigma, USA) were added to 100 μl of kidney homogenate, and the mixture was animated for 50 min. Then, 4 ml of 1-butanol (Sigma, USA) was added to the combination and vortexed 2 min through centrifugation at 5000 rpm for 15 min. The absorbance of the higher layer was measured at 532 nm (Spectro; Germany), and sequential concentrations of tetraethoxypropane (Sigma, USA) were used as the external standard. The antioxidant capacity of the kidney was measured using ferric reducing/antioxidant power (FRAP) assay. The FRAP substance consisted of 30 ml of acetate buffer (Sigma, USA) and 1.5 ml chloride ferric (Sigma, USA). In brief, 60 μl of kidney homogenate was added to 1.5 ml of newly prepared FRAP (Sigma, USA) in a test tube and incubated at 37°C for 10 min. The absorbance of the blue-colored complex was read against a blank at 593 nm. Sequential concentrations of FeSO4 · 7H2O (Sigma, USA) were used as an external standard.[1]

Griess technique

Griess technique was used to determine serum nitrite oxide (NO) level. Griess technique uses zinc sulfate powder to eliminate the serum protein of the samples. Accordingly, zinc sulfate powder (6 mg) was mixed with serum samples (400 μl) and vortexed for 1 min. The samples were centrifuged at 4°C for 10 min at 12,000 rpm, and the supernatant was used to measure the NO. Briefly, 50 μl of the sample was added to 100 μl of Griess reagent (Sigma; USA), and the reaction mixture was incubated for about 30 min at room temperature. The sample optical density was measured by ELISA reader (Hyperion; USA) at a wavelength of 450 nm according to manufacturer protocol.[13]

Statistical analysis

One-way analysis of variance was used for statistical analysis and Tukey post hoc test was applied to determine the difference between the groups. SPSS 16 (SPSS Corporation, Chicago, IL, USA) was used for data analysis, the results were expressed as a mean ± standard error, and P < 0.05 was considered statistically significant.

Results

Kidney function markers

Renal I/R increased significantly the mean BUN and creatinine concentration at the I/R group compared to the sham group (P < 0.05). The mean plasma BUN and creatinine concentration decreased significantly in RJ and RJ + I/R groups compared to the I/R group (P < 0.05). The mean BUN and creatinine concentration were not significant in RJ group compared to the sham group (P > 0.05) [Figure 1].

Subsequent renal I/R, lymphocytes were infiltrated significantly from renal vessels at the I/R group in comparison with the sham group (P < 0.05). The mean infiltrated lymphocytes could decrease significantly in animals treated with RJ in the RJ and RJ + I/R groups compared to the I/R group (P < 0.05). The lymphocytes infiltrated were not significant in RJ group compared to the sham group (P > 0.05) [Figure 2] and [Figure 3].

Figure 2: Mean of leukocyte infiltration per square millimeter, at the end of the reperfusion period, in renal ischemia/reperfusion and were treated with royal jelly. *: Significant different compared to the sham group (P < 0.05). &: Significant different compared to ischemia/reperfusion group (P < 0.05)

Morphometric analysis revealed that renal I/R decreased significantly the mean diameter of the glomerulus tubule in the I/R group compared to the sham group (P < 0.05). The diameter of the glomeruli increased significantly. In RJ and RJ + I/R groups compared to the I/R group (P < 0.05). The diameter of the glomerulus tubule was not significant in RJ group compared to the sham group (P > 0.05) [Figure 4].

The effect of renal I/R on ICAM-1 and TNF-α genes expression was increased significantly in the I/R group compared to the sham group (P < 0.05). RJ was able to decrease significantly the expression of both genes at RJ and RJ + I/R groups compared to the I/R group (P < 0.05). The ICAM-1 and TNF-α genes expression was not significant in RJ group compared to the sham group (P > 0.05) [Figure 5].

Figure 5: Results of real-time quantitative polymerase chain reaction on the tumor necrotic factor-alpha and intercellular adhesion molecule-1 mRNA expression in renal cortex of rats. Relative expression levels of each gene were obtained by using the comparative Ct method. *: A significant increase for ischemia/reperfusion group compared to the sham group (P < 0.05). &: A significant increase for intercellular adhesion molecule-1 mRNA expression at Royal Jelly group compared to ischemia/reperfusion group (P < 0.05).#: Significant increase for tumor necrotic factor-alpha mRNA expression at Royal Jelly treated group compared to ischemia/reperfusion group (P < 0.05)

The results of the testing oxidative stress in the groups showed that the kidney MDA level increased significantly in the renal I/R group compared to the sham group (P < 0.05). The kidney MDA level decreased significantly in RJ and RJ + I/R groups compared to the I/R group (P < 0.05). Similarly, renal I/R decreased the renal tissue FRAP level of the I/R group significantly compared to the sham group (P < 0.05). The administration of RJ increased the FRAP level in the kidney tissue significantly in RJ and RJ + I/R groups compared to the I/R group (P < 0.05) [Figure 6].

The mean NO in the blood serum increased significantly in the I/R group compared to the sham group (P < 0.05). The mean NO in the blood serum decreased significantly in RJ and RJ + I/R groups compared to the I/R group (P < 0.05). The mean NO was not significant in RJ group compared to the sham group (P > 0.05) [Figure 7].

Figure 7: Effects of Royal Jelly and ischemia/reperfusion on nitrite oxide level. *: Significant different compared to the sham group (P < 0.05). &: The significant difference compared to ischemia/reperfusion group (P < 0.05)

The absence of oxygen from blood during the I/R leads to oxidative injury through the induction of oxidative stress and inflammation.[8] The antioxidant activity of RJ inhibits oxidative stress and lipid peroxidation and protects DNA against oxidative stress.[5] The current study evaluated the effects of RJ in contradiction of renal I/R-induced damage in the rats. Along with the above issues should be noted, that in the present study, the I/R group was compared with sham group and I/R + RJ group was compared with I/R group. We found that the mRNA expression and lymphocyte infiltration of ICAM-1 and TNF-α increased significantly following I/R. This result is consistent with the results of Kelly et al. who showed that renal ischemia leads to incremental adjustments of adhesion molecules in the kidney.[14] The inflammatory responses to ischemia can be the main cause in the expansion of injuries. Especially, TNF-α through its direct destructive effects causes renal function impairment as well as decreasing blood flow and leukocytes infiltration.[15] Moreover, it triggers cell infiltration and creation of ICAM-1.[16] In endothelial cells, ICAM-1 gene expression increases within 1 h after renal I/R, which increases movement to the interstitium and leukocytes binding.[17] The cytokines cause the stimulation of the leukocytes after their translocation into the interstitium and exiting the blood vessels.[18] Conversely, the inhibition of the increase in ICAM-1 might protect kidneys in contrast to injuries caused by I/R.[19] In the present study, lymphocytes infiltrated into the interstitium subsequent renal I/R, which is in line with results of Rabb et al.[20] The decreased activation of lymphocyte and subsequent limit of their infiltration into the interstitium in the RJ treated groups is possibly due to the antioxidative effect of RJ and reduced expressions of TNF-α and ICAM-1 genes.[2] Aslan and Aksoy showed that the treatment of urinary tract with RJ reduced inflammatory cells.[21] In this regard, the results of Almeer et al., are consistent with the results of the present study, that RJ administration decreased significantly renal inflammation.[22] RJ seems to reduce TNF-α gene expression and reactive oxygen species (ROS) production.[23] It was suggested that the anti-inflammatory effects of RJ might be due to its content of flavonoids.[24] The results of the present study showed that the amount of NO increased significantly in the serum following I/R in I/R group. In RJ groups, there was a significant decrease in serum NO levels compared to I/R group. NO is an important molecule that plays a key role in body physiology.[25] NO molecule produced by inducible NO synthase (iNOS) can induce DNA damage and structural degradation of many lipids and proteins.[1] I/R seems to cause cell damage by up-regulation of iNOS.[26] The results of Ferdinandy and Schulz were consistent with the results of the present study, suggesting that I/R could increase significantly the expression of iNOS.[27] RJ can inhibit NO inducing lipopolysaccharide by inducing the expression of Heme oxygenase-1 and calmodulin/calcium-dependent protein kinase-4.[28] The results of the current study also showed that RJ is able to reduce lipid peroxidation and increase the antioxidant capacity of kidney tissue, thus reducing oxidative stress. Accordingly, it appears that RJ with its anti-oxidant properties could reduce MDA and increase FRAP in the treatment groups by inhibiting the production of ROS. The results of Silici et al. study were consistent with the results of this study, which showed that RJ could reduce MDA levels.[29] In this study, a 30-min ischemia and 24-h reperfusion led to an increase in plasma creatinine and BUN concentrations at I/R group compared to the sham group. RJ at the administrated dose was capable to decrease the level of creatinine and BUN. These results suggest that the growth in creatinine concentration by I/R is accompanied by the decrease in glomerular filtration rate.[30] Renal creatinine clearance is the indicator of glomerular filtration rate and decreases following the I/R. One of the main reasons for the reduction in renal blood flow subsequent I/R is the trouble of the equilibrium among the vasodilator (such as NO).[31] These results are in agreement with those of Momeni et al. who found that RJ reduces NO in rat's testis, probably by iNOS inhibition.[24] Therefore, this information recommends that RJ, possibly through accumulative blood flow, decreasing cell damages, and reducing the pressure of Bowman's space, leading to the enhanced glomerular filtration rate and improvement of the BUN and creatinine levels.[22] In addition, a part of the protecting effects of RJ can originate from the reduction in TNF-α construction, which leads to enhanced renal blood flow and glomerular filtration rate.[23] The treatment of animals with RJ in this study increased the diameter compared to the I/R group. A rupture in the glomerular order diminished glomerular size and damage of proximal tubules, which are some of the changes that can indicate the pathological effects of I/R on glomeruli.[32] It seems that a decrease in the mean diameter of glomeruli can be associated with kidney functional disorders.[1] RJ can reduce platelet aggregation, lower fibrinogen and have direct effects on inflammatory.[33]

RJ seems to be able to reduce oxidative stress. Therefore, according to the results of the present study, it neutralizes the effects of I/R.

Briefly, the current study showed that RJ contributes to reducing oxidative stress and inflammation due to both 30 min renal ischemia and 24 h reperfusion in the animals studied. Furthermore, RJ can recover the renal damage subsequent from I/R, possibly through its antioxidant properties, decreasing the activation of pro-inflammatory elements, and correcting the blood flow.